Abstract

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1-5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes (ompFW) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture.

Real-time PCR results of Escherichia coli cells exposed to BC at 30 and 60 min, and at OD 0.1 and 0.5 after inoculation (equivalent to sampling points 1, 2, 3 and 4, respectively, shown in ). The expression is presented as log 2 of fold change compared to the reference gene, accD, and the control. The expression was based on 3 biological replicates.

Real-time PCR results from a competitive growth experiment of mixed cultures of knock-out strain b1171 and the wild type, and knock-out strain ybjX and the wild type. The time points used were equivalent to sampling points 1, 2, 3 and 4 shown in . The log 2 of fold change was calculated using the ΔΔCT calculation based on gene accD and the kanamycin resistance gene related to the growth in the presence and absence of BC. The expression was based on 3 biological replicates.